The present invention relates to methods for providing tomato plants with increased levels of acylsugars, methods for controlling pests in tomato, and tomato plants with acylsugars. All publications cited in this application are herein incorporated by reference.
Fruit and vegetable plants are attacked by a variety of insect pests that cause losses directly through feeding on leaves, flowers, fruit, and vegetables, and indirectly through the transmission of viruses, resulting in reduction in yield and fruit/vegetable quality. Pest control products are becoming more expensive and narrower in spectrum, which could result in the need for more sprays or the use of combinations of chemicals. In addition, the loss of label can eliminate the use of some pest control products for certain fruit or vegetable plants. Alternative methods of pest control are needed.
The development of sustainable, environmentally benign methods of crop protection is an important priority in agricultural research. A variety of insects attack crops, causing damage and reducing yields and crop quality. Insects cause crop loss directly through feeding on leaves, flowers, fruit, or seed. A subset of insects damages crops indirectly, through transmission of plant viruses, resulting in reduced yield and crop quality. Breeding for disease resistance has been an important strategy for protection of crops against fungal, bacterial, or viral diseases.
Although integrated pest management (IPM) strategies have been implemented with noted success, insect control has more often relied on the use of pesticides, leading to the evolution of pesticide-resistant insects and to increasing health and environmental concerns. The development of pest resistant plants is an attractive alternative strategy for the control of insects and the direct damage they cause.
Host/insect interactions for plant protection were originally classified as being due to antibiosis, nonpreference, or tolerance (Painter, 1958; Beck, 1965), although the term “antixenosis” was suggested as a more accurate term than nonpreference (Kogan and Ortman, 1978). Under antibiosis a resistant plant exerts an adverse effect on the growth and survival of the insect. Antibiosis can be due to physical characteristics of the plant or due to secondary metabolites such as toxins. Under antixenosis (non-preference), a plant exerts influences on insect behavior, deterring the insect from using the plant as a host (Painter, 1958; Beck, 1965), hence the use of the term “deterrence” in some references. “Tolerance” indicates that the pest is neither deterred from the host plant nor adversely affected by the host plant, but the damage resulting from the pest infestation is reduced compared to that suffered by susceptible varieties of the crop (Painter, 1958; Beck, 1965; Reese et al., 1994). These systems of insect resistance may not be mutually exclusive. It is possible that a resistance mechanism could have aspects of both antibiosis and deterrence.
Breeding for insect resistance has a long history, although insect resistance has been used less than disease resistance in most crops. The wheat variety “Underhill” was reported to have Hessian fly resistance in 1782. Despite resistance breakdown over the years in a number of Hessian fly resistance sources, many wheat varieties have been bred to include this trait (Panda and Khush, 1995; Everson and Gallun, 1980). Another historical example is grape phylloxera (Daktulosphaira vitifoliae), a North American aphid that was inadvertently transferred to France about 1860. Grape phylloxera feeds on grape roots, resulting in decreased productivity and vine death. Wild North American grape possessed natural resistance to the pest. This resistance was transferred to develop phylloxera resistant rootstocks that saved the French wine industry. Rootstocks with similar resistance are still in use (Granett et al., 2001).
Some systems of natural insect resistance are based upon physical structures or characteristics. A resistance to potato leafhopper (Empoasca fabae) in bean (Phaseolus vulgaris) is due to a high density of hooked nonglandular trichomes. These trichomes act as physical barriers, entrapping nymphs as their hooks become embedded in the nymphs' bodies (Pillemer and Tingey, 1976, 1978). The waxy surface of plants has also been implicated in reducing insect infestation. “Glossy” mutants, lacking the normal waxy layer or “bloom” of non-mutant plants, have been found in a number of crop species. Sadasivan and Thayumanavan (2003) list instances in Brassica, raspberry, castor, sorghum, wheat, sugarcane, and onion in which the glossy plants are more susceptible to a variety of insect pests than the normal waxy plants. This could be due to adverse effects of the waxy layer on the ability of insects to adhere, move, or feed on the plant. Differences in wax layer may also affect the choice of the plant for feeding or oviposition. Consequently, such waxy surfaces may confer either antibiosis or antixenosis depending on their mode of action against different pests.
A number of insect resistance systems are based upon secondary metabolites that are toxic or otherwise detrimental or noxious to pests. Secondary metabolites are a very diverse array of compounds that are produced by plants but which are not considered essential for basic metabolic function or processes. There are too many secondary metabolites to describe in any detail here (see Hadacek, 2002; Singer et al., 2003; Sadasivan and Thayumanavan, 2003), but a few well-known examples are 2-tridecanone, cucurbitacins, and glycoalkaloids.
The 2-tridecanone, a methyl ketone, is a secondary metabolite in glandular trichomes that is the basis of insect resistance in Lycopersicon hirsutum var. glabratum (Williams et al., 1980; Fery and Kennedy, 1987). 2-tridecanone has been implicated in the resistance of L. hirsutum to tobacco hornworm (Manduca sexta), spider mite species (Tetranychus spp.), Colorado potato beetle (Leptinotarsa decemlineata), tomato pinworm (Keiferia lycopersicella), and beet armyworm (Spodoptera exigua) (Kennedy, 1976; Gonyalves et al., 1998; Farrar and Kennedy, 1991; Lin et al., 1987; Maluf et al., 1997). This compound is quite toxic, and also acts as an oviposition and/or feeding deterrent.
The plant species L. pennellii Corr. is a wild relative of the cultivated tomato, L. esculentum. As a plant species, L. pennellii is morphologically intermediate between potato and tomato. However, since L. pennellii is interfertile in controlled pollinations with the cultivated tomato, it is commonly grouped with other wild species of tomato.
Physical entrapment of arthropods by the exudate from glandular hairs of various plants is known in wild Solanum species such as S. berthaultii, S. tarijense, and S. polyadenium. The exudate of the four-lobed (type A) trichomes, when exposed to atmospheric oxygen, forms a viscous substance which accumulates on the tarsi and mouthparts of green peach aphid (Myzus persicae Sulzer), the potato aphid (Macrosiphum euphorbiae Thomas), and the potato leafhopper (Empoasca fabae Harris). The viscous material hardens and effectively immobilizes the insects, resulting in their death through starvation. S. berthaultii also possesses a second type of glandular trichome (type B) which is slender and continuously secretes a sticky substance at its tip. This type of trichome has been found to be important in entrapping the two-spotted spider mite (Tetranychus urticae Koch) and tarsonemid mites. Mites are not powerful enough to rupture the membrane of the four-lobed glandular trichomes. Utilizing an electronic feeding monitor, Lapointe and Tingey (1984) demonstrated that aphid feeding on S. berthaultii leaves was characterized by a delay in probing, a decrease in the duration of probes, and that an overall physical removal of the type B exudate resulted in a decrease of resistance as measured by these parameters.
The most abundant of the types of glandular hairs in the genus Lycopersicon are the type IV and VI trichomes. The type VI trichome is similar in appearance to the type A trichomes on Solanum species while the type IV trichome is similar to the type B of Solanum. Physical entrapment of the carmine spider mite (Tetranychus cinnabarinus Boisduval), the two-spotted spider mite (T. urticae), and the greenhouse whitefly (Trialeurodes vaporariorum Westwood) by type IV glandular exudate appears to be the principal component of resistance to these pests by certain Lycopersicon species (Gentile et al., 1969, 1968). Removal of the exudate with alcohol resulted in successful oviposition and normal nymphal development of the greenhouse whitefly (Gentile et al., 1968). The release of a viscous exudate upon rupture of the type VI trichomes is suggested as the basis for physical entrapment of insects in several wild tomato species.
L. pennellii, especially accession LA716, is resistant to several insect species, including greenhouse whitefly, carmine and two-spotted spider mites, and potato and green peach aphids. Insect resistance in L. pennellii is attributed to the type IV glandular hairs, which are not present on the foliage of L. esculentum. Resistance to greenhouse whitefly has been attributed to the entrapment of adults in the sticky exudate of type IV trichomes (Gentile et al., 1968). Physical entrapment of carmine and two-spotted spider mites and potato aphids in exudate of type IV trichomes was also suggested as the mode of resistance to these pests (Gentile et al., 1969, Gentile and Stoner, 1968b). Clayberg (1975) observed that a periclinal chimera, consisting of the epidermis, with dense indumentum of L. pennellii and a “core” of L. esculentum origin, had levels of whitefly resistance equal to that in L. pennellii but a reduced level of resistance to potato aphids.
The type IV trichome of L. pennellii, its hybrids and progeny are slender hairs with pointed tips about 0.2 mm to 0.4 mm in length, standing on a large simple basal cell. The hair is glandular and it continuously secretes a droplet which is not membrane-bound. Further details of these trichomes may be found in Luckwill (1943). The exudate of the type IV trichomes of L. pennellii, its hybrids, and progeny is composed of a complex mixture of glucose triesters of saturated straight chain and branched fatty acids (Burke et al., 1987). The most abundant fatty acids found in L. pennellii glucose esters include 2-methylpropanoic, 8-methylnonanoic, and n-decanoic acids, with 2-methylbutanoic, 3-methylbutanoic, and n-dodecanoic acids being present in relatively minor amounts. The positions of esterification have all been found to be the 2, 3, and 4 positions.
Choice of breeding or selection methods depends on the mode of plant reproduction, the heritability of the trait(s) being improved, and the type of cultivar used commercially (e.g., F1 hybrid cultivar, pureline cultivar, etc.). For highly heritable traits, a choice of superior individual plants evaluated at a single location will be effective, whereas for traits with low heritability, selection should be based on mean values obtained from replicated evaluations of families of related plants. Popular selection methods commonly include pedigree selection, modified pedigree selection, mass selection, and recurrent selection.
The complexity of inheritance influences choice of the breeding method. Backcross breeding is used to transfer one or a few favorable genes for a highly heritable trait into a desirable cultivar. This approach has been used extensively for breeding disease-resistant cultivars. Various recurrent selection techniques are used to improve quantitatively inherited traits controlled by numerous genes. The use of recurrent selection in self-pollinating crops depends on the ease of pollination, the frequency of successful hybrids from each pollination and the number of hybrid offspring from each successful cross.
Each breeding program should include a periodic, objective evaluation of the efficiency of the breeding procedure. Evaluation criteria vary depending on the goal and objectives, but should include gain from selection per year based on comparisons to an appropriate standard, overall value of the advanced breeding lines, and number of successful cultivars produced per unit of input (e.g., per year, per dollar expended, etc.).
Promising advanced breeding lines are thoroughly tested and compared to appropriate standards in environments representative of the commercial target area(s) for three or more years. The best lines are candidates for new commercial cultivars; those still deficient in a few traits may be used as parents to produce new populations for further selection.
These processes, which lead to the final step of marketing and distribution, usually take from eight to twelve years from the time the first cross is made. Therefore, development of new cultivars is a time-consuming process that requires precise forward planning, efficient use of resources, and a minimum of changes in direction.
A most difficult task is the identification of individuals that are genetically superior, because for most traits the true genotypic value is masked by other confounding plant traits or environmental factors. One method of identifying a superior plant is to observe its performance relative to other experimental plants and to a widely grown standard cultivar. If a single observation is inconclusive, replicated observations provide a better estimate of its genetic worth.
The development of new tomato cultivars requires the development and selection of tomato varieties, the crossing of these varieties and selection of superior hybrid crosses. The hybrid seed is produced by manual crosses between selected male-fertile parents or by using male sterility systems. These hybrids are selected for certain single gene traits such as pod color, flower color, pubescence color or herbicide resistance which indicate that the seed is truly a hybrid. Additional data on parental lines, as well as the phenotype of the hybrid, influence the breeder's decision whether to continue with the specific hybrid cross.
Pedigree breeding and recurrent selection breeding methods are used to develop cultivars from breeding populations. Breeding programs combine desirable traits from two or more cultivars or various broad-based sources into breeding pools from which cultivars are developed by selfing and selection of desired phenotypes. The new cultivars are evaluated to determine which have commercial potential.
Pedigree breeding is used commonly for the improvement of self-pollinating crops. Two parents that possess favorable, complementary traits are crossed to produce an F1. An F2 population is produced by selfing one or several F1s. Selection of the best individuals may begin in the F2 population; then, beginning in the F3, the best individuals in the best families are selected. Replicated testing of families can begin in the F4 generation to improve the effectiveness of selection for traits with low heritability. At an advanced stage of inbreeding (i.e., F6 and F7), the best lines or mixtures of phenotypically similar lines are tested for potential release as new cultivars.
Mass and recurrent selections can be used to improve populations of either self- or cross-pollinating crops. A genetically variable population of heterozygous individuals is either identified, or created, by intercrossing several different parents. The best plants are selected based on individual superiority, outstanding progeny, or excellent combining ability. The selected plants are intercrossed to produce a new population in which further cycles of selection are continued.
Backcross breeding has been used to transfer genes for a simply inherited, highly heritable trait into a desirable homozygous cultivar or inbred line which is the recurrent parent. The source of the trait to be transferred is called the donor parent. After the initial cross, individuals possessing the phenotype of the donor parent are selected and repeatedly crossed (backcrossed) to the recurrent parent. The resulting plant is expected to have the attributes of the recurrent parent (e.g., cultivar) and the desirable trait transferred from the donor parent.
The single-seed descent procedure in the strict sense refers to planting a segregating population, harvesting a sample of one seed per plant, and using the one-seed sample to plant the next generation. When the population has been advanced from the F2 to the desired level of inbreeding, the plants from which lines are derived will each trace to different F2 individuals. The number of plants in a population declines each generation due to failure of some seeds to germinate or some plants to produce at least one seed. As a result, not all of the F2 plants originally sampled in the population will be represented by a progeny when generation advance is completed.
Having a morphology that is intermediate between potato and tomato means that L. pennelli is horticulturally unsuitable for either home or commercial production of tomatoes. Cultivated tomatoes lack the high concentration and types of acylsugars of L. pennellii and therefore lack the multiple pest resistance of L. pennellii. 